Attenuated, genetically-engineered pseudorabies virus S-PRV-155 and uses thereof

ABSTRACT

The present invention provides an attenuated, genetically-engineered pseudorabies virus designated S-PRY-155 (ATCC Accession No. VR 2311). A vaccine is provided which comprises an effective immunizing amount of S-PRY-155 and a suitable carrier. A method of immunizing an animal against disease caused by pseudorabies virus is also provided which comprises administering to the animal an effective immunizing dose of the vaccine. The present invention also provides a method for distinguishing an animal vaccinated with the vaccine of the present invention from an animal infected with a naturally-occurring, wild-type pseudorabies virus.

This application is a divisional application of U.S. Ser. No. 663,413,filed Mar. 1, 1991, now U.S. Pat. No. 5,240,703.

Within this application several publications are referenced by arabicnumerals within parentheses. Full citations for these references may befound at the end of the specification immediately preceding the claims.The disclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

FIELD OF THE INVENTION

The present invention involves an attenuated pseudorabies virus usefulin a live virus vaccine to protect swine from pseudorabies. Theinvention further involves the use of such a virus to permit one todistinguish between an animal which has been vaccinated with the virusand an animal infected by wild-type pseudorabies virus.

BACKGROUND OF THE INVENTION

The ability to isolate viral DNA and clone this isolated DNA intobacterial plasmids has greatly expanded the approaches available to makeviral vaccines. The methods used to make the present invention involvemodifying cloned vital DNA sequences by insertions, deletions and singleor multiple base changes. The modified DNA is then reinserted into theviral genome to render the virus non-pathogenic. The resulting livevirus may then be used in a vaccine to elicit an immune response in ahost animal and to protect the animal against a disease.

One group of animal viruses, the herpesviruses or Herpetoviridae, is anexample of a class of viruses amenable to this approach. These virusescontain 100,000 to 200,000 base pairs of DNA as their genetic material.Importantly, several regions of the genome have been identified that arenonessential for the replication of virus in vitro in cell culture.Modifications in these regions of the DNA may lower the pathogenicity ofthe virus, i.e., attenuate the virus. For example, inactivation of thethymidine kinase gene renders human herpes simplex virus non-pathogenic(13), and pseudorabies virus of swine non-pathogenic (14).

Removal of part of the repeat region renders human herpes simplex virusnon-pathogenic (16,17). A repeat region has been identified in Marek'sdisease virus that is associated with viral oncogenicity (18). A regionin herpesvirus saimiri has similarly been correlated with oncogenicity(19). Removal of part of the repeat region renders pseudorabies virusnon-pathogenic (U.S. Pat. No. 4,877,737, issued Oct. 31, 1989). A regionin pseudorabies virus has been shown to be deleted innaturally-occurring vaccine strains (7,15) and it has been shown thatthese deletions are at least partly responsible for the lack ofpathogenicity of these strains.

It is generally agreed that herpesviruses contain non-essential regionsof DNA in various parts of the genome, and that modifications of theseregions can attenuate the virus, leading to a non-pathogenic strain fromwhich a vaccine may be derived. The degree of attenuation of the virusis important to the utility of the virus as a vaccine. Deletions whichcause too much attenuation of the virus will result in a vaccine thatfails to elicit an adequate immune response. Although several examplesof attenuating deletions are known, the appropriate combination ofdeletions is not readily apparent.

The natural host of pseudorabies virus is swine, in which infection iscommonly not apparent but may be characterized by fever, convulsions andparalysis. Pseudorabies virus also infects cattle, sheep, dogs, cats,foxes and mink, where infection usually results in death of the host.The predominant visible feature of pseudorabies viral infection isintense pruritus generally resulting in host mutilation of the involvedarea. Violent excitement, fits and paralysis, all symptoms ofencephalomyelitis, precede death which usually occurs within a few daysfollowing onset of clinical signs.

Pseudorabies virus disease in swine is of serious concern togovernmental bodies worldwide. In the United States, swine from infectedherds cannot be sold except to slaughterhouses. The U.S. Department ofAgriculture has enacted an eradication program to eliminatepseudorabies. Prior to the development of specific differential vaccinesand companion diagnostic tests, any animal vaccinated for pseudorabieswas treated as though it were infected and was subjected to the sameregulatory constraints. With the advent of differential vaccines,regulations have been modified to allow interstate shipment ofvaccinated, non-infected swine provided the differentialvaccine/diagnostic test combination has been approved for use in theCooperative State-Federal Pseudorabies Eradication Program (FederalRegister, Vol. 55, No. 90, pp. 19245-19253 (May 9, 1990)).

The construction of differential vaccines has focused on the deletion ofone of the glycoproteins of PRV. Theoretically, the glycoprotein chosento be the diagnostic marker should have the following characteristics:(1) the glycoprotein and its gene should be non-essential for theproduction of infectious virus; (2) the glycoprotein should elicit astrong and persistent response in the animal; (3) the glycoproteinshould not make a contribution to the protective immune response. Threeglycoproteins gD, gH, and gB(II) (25) fail the first criteria. Each hasa counterpart in herpes simplex virus (HSV) which has proven to beessential (20). g63 is a minor glycoprotein and would not be expected toelicit a major serological response (8). gIII has been shown to make asignificant contribution to protective immunity as a target ofneutralizing antibodies (10) and as a target of cell-mediated immunity(21). gI is also the target of neutralizing antibodies (22) and might beexpected to play a role in protective immunity. Only gpX meets all threeof the theoretical criteria (25).

Currently, there are five USDA-licensed, modified live, differential PRVvaccines available. They are PRV/MARKER® (SyntroVet Incorporated),TOLVID® (Upjohn), PRV vaccine (Boehringer Ingelheim), PRVAC® (SmithKlineBeecham Animal Health) and OMNIMARK™ (Fermenta Animal Health). Thecompanion diagnostic test for each of these vaccines is directed againstone of three PRY glycoproteins (gpX, gI or gIII). In each case, the genecoding for the diagnostic glycoprotein has either been altered bygenetic engineering or has been shown to be deleted in a naturallyoccurring virus. The diagnostic marker deleted from PRY/Marker andTolvid is gpX, which was altered by genetic engineering techniques. PRVvaccine and PRVac contain viruses that have a spontaneous deletion of gIfrom a naturally occurring virus. gIII is deleted from OmniMark, as aresult of genetic engineering.

As the pseudorabies eradication program progresses, it would be of greatvalue to have a confirmatory diagnostic test. Due to differingantigenicity of each diagnostic antigen and to the nature of the immuneresponse of individual animals, the level of antibody to the diagnosticantigen can vary widely. A vaccine which incorporates a seconddiagnostic marker which could be used in a confirmatory test would be ofgreat value. Two virus strains have been described which haveincorporated the deletion of two glycoproteins for the purposes ofserologic differentiation. One of these, described by Kit et al. (U.S.Pat. No. 4,711,850), has a genetically engineered deletion of gIII and anaturally occurring deletion of gI. As discussed above, both of theseglycoproteins are targets of neutralizing antibody and gIII is also thetarget of cell-mediated immunity. The deletion of both of theseimportant antigens would be expected to compromise the efficacy of avaccine. The second virus, described by Post et al. (25) has beengenetically engineered to incorporate deletions in both the gpX and gIgenes. The authors concluded that this virus was significantlycompromised in efficacy relative to a virus in which only the gpX wasdeleted. In summary, the current state of the art indicates that a virusdeleted in both gpX and gI would not be effective as a vaccine.

A vaccine superior to the currently available products would have thefollowing characteristics: (1) not produce clinical signs in 3-4 day oldpiglets; (2) give 95% protection in pigs of all ages; (3) permitserological differentiation from wild-type infected animals: and (4)permit a confirmatory diagnostic test. This invention provides such asuperior vaccine, unexpectedly, by deleting specific regions of both gpXand gI.

SUMMARY OF THE INVENTION

Specifically, the present invention provides an attenuated,genetically-engineered pseudorabies virus designated S-PRV-155 (ATCCAccession No. VR 2311). The present invention also provides a vaccinewhich comprises an effective immunizing amount of the attenuated,genetically-engineered pseudorabies virus designated S-PRV-155 and asuitable carrier. The invention further provides a method of immunizingan animal against disease caused by pseudorabies virus which comprisesadministering to the animal an effective immunizing dose of the vaccineof the present invention. Finally, the invention provides a method fordistinguishing an animal vaccinated with the vaccine of the presentinvention from an animal infected with a naturally-occurring, wild-typepseudorabies virus.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Details of the PRV Strain ISU S62/26. Diagram of PRV genomic DNAshowing the unique long, internal repeat, unique short, and terminalrepeat regions. Restriction maps for the enzymes BamHI, XbaI, andHindIII are indicated. Fragments are numbered or lettered in order ofdecreasing size. The unique short region is also expanded for inclusionof more detail. The location of several genes is also indicated; theyare glycoprotein II (gII) (24), glycoprotein III (gIII) (23), thymidinekinase (TK), immediate early gene (IE), protein kinase (PK) (3),glycoprotein X (gpX) (4), glycoprotein 50 (g50) (5), glycoprotein 63(g63) (6), and glycoprotein I (gI) (6).

FIGS. 2A-2D. Detailed description of the DNA insertion in HomologyVector 263-58.18. Diagram showing the orientation of DNA fragmentsassembled in plasmid 263-58.18. The origin of each fragment is indicatedin the table. The sequences located at each of the junctions betweenfragments is also shown. The restriction sites used to generate eachfragment as well as synthetic linker sequences which were used to Jointhe fragments are described for each Junction. The synthetic linkersequences are underlined by a heavy bar. The location of several genecoding regions and regulatory elements is also given. The followingconventions are used: numbers in parentheses, (), refer to amino acidsrelative to the references given in FIG. 1, and restriction sites inbrackets, [], indicate the remnants of sites which were destroyed duringconstruction.

The following abbreviations are used: protein kinase (PK), glycoproteinX (gpX), glycoprotein 50 (g50), glycoprotein 63 (g63), pseudorabiesvirus (PRV), and polyadenylation signal (pA).

FIGS. 3A-3D. Detailed description of the DNA insertion in HomologyVector 416-09.2H. Diagram showing the orientation of DNA fragmentsassembled in plasmid 416-09.2H. The origin of each fragment is indicatedin the table. The sequences located at each of the junctions betweenfragments is also shown. The restriction sites used to generate eachfragment as well as synthetic linker sequences which were used to jointhe fragments are described for each junction. The synthetic linkersequences are underlined by a heavy bar. The location of several genecoding regions and regulatory elements is also given. The followingconventions are used: numbers in parentheses, (), refer to amino acidsrelative to the references given in FIG. 1, and restriction sites inbrackets, [], indicate the remnants of sites which were destroyed duringconstruction. The following abbreviations are used: protein kinase (PK),glycoprotein X (gpX), glycoprotein 50 (g50), glycoprotein 63 (g63),pseudorabies virus (PRV), and polyadenylation signal (pA).

FIGS. 4A-4D. Detailed description of the DNA insertion in HomologyVector 436-86.32K. Diagram showing the orientation of DNA fragmentsassembled in plasmid 436-86.32K. The origin of each fragment isindicated in the table. The sequences located at each of the junctionsbetween fragments is also shown. The restriction sites used to generateeach fragment as well as synthetic linker sequences which were used tojoin the fragments are described for each junction. The synthetic linkersequences are underlined by a heavy bar. The location of several genecoding regions and regulatory elements is also given. The followingconventions are used: numbers in parentheses, (), refer to amino acidsrelative to the references given in FIG. 1, and restriction sites inbrackets, [], indicate the remnants of sites which were destroyed duringconstruction. The following abbreviations are used: protein kinase (PK),glycoprotein X (gpX), glycoprotein 50 (g50), glycoprotein I (gI),pseudorabies virus (PRV), human cytomegalovirus (HCMV), andpolyadenylation signal (pA).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides an attenuated, genetically-engineeredpseudorabies virus designated S-PRV-155 (ATCC Accession No. VR 2311).The S-PRV-155 pseudorabies virus has been deposited pursuant to theBudapest Treaty on the International Deposit of Microorganisms for thePurposes of Patent Procedure with the Patent Culture Depository of theAmerican Type Culture Collection, 12301 Parklawn Drive, Rockville, Md.20852 U.S.A. under ATCC Accession No. VR 2311. The present inventionalso provides a vaccine which comprises an effective immunizing amountof the attenuated, genetically-engineered pseudorabies virus designatedS-PRV-155 and a suitable carrier. The vaccine may contain eitherinactivated or live pseudorabies virus S-PRV-155.

Suitable carriers for the pseudorabies virus are well known in the artand include proteins, sugars, etc. One example of such a suitablecarrier is a physiologically balanced culture medium containing one ormore stabilizing agents such as stabilized, hydrolyzed proteins,lactose, etc.

In general, the vaccine of this invention contains an effectiveimmunizing amount of S-PRV-155 virus of from about 10³ to 10⁹ PFU/dose.Preferably, the effective immunizing amount is from about 10⁴ to 10⁶PFU/dose for the live vaccine and from about 10⁷ to 10⁹ PFU/dose for theinactivated vaccine. Preferably, the live vaccine is created by takingtissue culture fluids and adding stabilizing agents such as stabilized,hydrolyzed proteins. Preferably, the inactivated vaccine uses tissueculture fluids directly after inactivation of the virus.

The present invention also provides a method of immunizing an animal,particularly a swine, against disease caused by pseudorabies virus whichcomprises administering to the animal an effective immunizing dose ofthe vaccine of the present invention. The vaccine may be administered byany of the methods well known to those skilled in the art, for example,by intramuscular, subcutaneous, intraperitoneal or intravenousinjection. Alternatively, the vaccine may be administered intranasallyor orally.

The present invention also provides a method for distinguishing ananimal vaccinated with the vaccine of the present invention from ananimal infected with a naturally-occurring, wild-type pseudorabiesvirus. This method comprises analyzing a sample of a body fluid from theanimal for the presence of gpX and at least one other antigen normallyexpressed in an animal infected by a naturally-occurring, wild-typepseudorabies virus and determining whether the antigen and gpX arepresent in the body fluid. The presence of the antigen and the absenceof gpX in the body fluid is indicative of an animal vaccinated with thevaccine and not infected with a naturally-occurring, wild-typepseudorabies virus. The presence of the antigen and of gpX in the bodyfluid may be determined by various methods, for example, by detecting inthe body fluid antibodies specific for the antigen and for gpX. Themethod for distinguishing an animal vaccinated with the vaccine of thepresent invention from an animal infected with a naturally-occurring,wild-type pseudorabies virus may further comprise analyzing the samplefor the presence of gI. The presence of the antigen and the absence ofgI in the body fluid is indicative of an animal vaccinated with thevaccine and not infected with a naturally-occurring, wild-typepseudorabies virus. Within this application, a naturally-occurringpseudorabies virus means a pseudorabies virus which has not beengenetically engineered, and includes, but is not limited to, wild-typepseudorabies viruses and pseudorabies viruses selected from pseudorabiesviruses which exist in nature and have spontaneous deletions. Withinthis application, nonessential gene means a gene which is not essentialfor viral replication.

The present invention also provides a further method for distinguishingan animal vaccinated with the vaccine of the present invention from ananimal infected with a naturally-occurring, wild-type pseudorabiesvirus. This method comprises analyzing a sample of a body fluid from theanimal for the presence of gI and at least one other antigen normallyexpressed in an animal infected by a naturally-occurring, wild-typepseudorabies virus and determining whether the antigen and gI arepresent in the body fluid. The presence of the antigen and the absenceof gI in the body fluid is indicative of an animal vaccinated with thevaccine and not infected with a naturally-occurring, wild-typepseudorabies virus.

Methods for constructing, selecting and purifying pseudorabies viruses,including S-PRV-155, are detailed in the Materials and Methods sectionwhich follows.

MATERIALS AND METHODS

PREPARATION OF PSEUDORABIES VIRUS (PRV) STOCK SAMPLES. Pseudorabiesvirus (PRY) stock samples were prepared by infecting Vero cells at amultiplicity of infection of 0.01 plaque forming units (PFU)/cell inDulbecco's Modified Eagle (DME) medium containing 2 mM glutamine, 100units/ml penicillin and 100 units/ml streptomycin (these components wereobtained from Irvine Scientific or an equivalent supplier, and hereafterare referred to as complete DME medium) plus 1% fetal bovine serum.After cytopathic effect was complete, the medium and cells wereharvested and the cells were pelleted at 3000 rpm for 5 minutes in aclinical centrifuge. The cells were resuspended in 1/10 the originalvolume of medium and an equal volume of two times autoclaved skim milk(9% skim milk powder in H₂ O wgt/vol) was added. The virus sample wasfrozen and thawed two times, aliquoted and stored frozen at -70° C. Thetiter was usually about 10⁸ PFU/ml.

TITRATION OF PRV. For PRV titrations, Vero cells were plated in 60 mmpetri dishes at 5×10⁵ cells/mi. The next day growth medium (complete DMEplus 10% fetal bovine serum) was replaced with 3.0 ml/dish ofmaintenance medium (complete DME plus 1% fetal bovine serum). Virusstocks were diluted 1:10⁴, 10⁵, 10⁶, 10⁷, and 10⁸ in maintenance medium.From these dilutions, 1.0 ml was added to each dish. Dishes were set at37° C. in a humidified incubator with 5% CO₂ for 4 hours. The media wasthen aspirated and each dish was overlaid with 5.0 ml 0.75% finalconcentration of low melting point agarose in 2X Minimal Essential Media(MEM) containing 2 mM glutamine, 100 units/ml penicillin, 100 units/mlstreptomycin, and 2% fetal bovine serum. Dishes were set at roomremperature for 30 minutes, to let the agarose set, and then placed at37° C. in a humidified incubator with 5% CO₂ for 3 days. Plaques werecounted and PFU/ml calculated.

PREPARATION OF PRV DNA. For PRV DNA preparation a confluent monolayer ofVero cells in a 25 cm² flask or a 60 mm petri dish was infected with 100microliters of virus sample in 1 ml medium. Adsorption proceeded for 1-2hours at 37° C. in a humidified incubator with 5% CO₂ in air. Afteradsorption, 4 mls of complete DME medium plus 1% fetal bovine serum wereadded. After overnight incubation, or when the cells were showing 100%cytopathic effect, the cells were scraped into the medium with a cellscraper (Costar brand). The cells and medium were centrifuged at 3000rpm for 5 minutes in a clinical centrifuge. The medium was decanted andthe cell pellet was gently resuspended in a 0.5 ml solution containing0.01M Tris pH 7.5, 1 mM EDTA and 0.5% Nonidet P-40 (NP40). The samplewas incubated at room temperature for 10 minutes. Ten microliters of astock solution of RNase A (Sigma) were added (stock is 10 mg/ml, boiledfor 10 minutes to inactivate DNase). The sample was centrifuged for 5minutes at 3000 rpm in a clinical centrifuge to pellet nuclei. The DNApellet was removed with a pasteur pipette or wooden stick and discarded.The supernatant fluid was decanted into a 1.5 ml Eppendorf tubecontaining 25 microliters of 20% sodium dodecyl sulfate (Sigma) and 25microliters proteinase-K (10 mg/ml; Boehringer Mannhiem). The sample wasmixed and incubated at 37° C. for 30-60 minutes. An equal volume ofwater-saturated phenol was added and the sample was mixed on a vortexmixer. The sample was centrifuged in an Eppendorf minifuge for 5 minutesat full speed. The upper aqueous phase was removed to a new Eppendorftube, two volumes of -20° C. absolute ethanol were added and the tubewas put at -20° C. for 30 minutes to precipitate nucleic acid. Thesample was centrifuged in an Eppendorf centrifuge at 4° C. for 5minutes. The supernatant was decanted and the pellet was washed one timewith cold 80% ethanol. The pellet was rehydrated in 17 microliters ofwater. For the preparation of larger amounts of DNA, the procedure wasscaled up to start with an 850 cm² roller bottle of Vero cells. The DNAwas stored in water or 0.01M Tris pH 7.5, 1 mM EDTA at 4° C.

PHENOL EXTRACTION. Phenol extraction was performed on any convenientvolume of DNA sample, typically between 100 microliters to 1 ml. The DNAsample was diluted in 0.01M Tris pH 7.5, 1 mM EDTA and an equal volumeof water saturated phenol was added. The sample was mixed briefly on avortex mixer and placed on ice for 3 minutes. After centrifugation for 3minutes in a microfuge, the aqueous layer was removed to a new tube andwas precipitated by ethanol.

ETHANOL PRECIPITATION. DNA in a sample was concentrated by ethanolprecipitation. To the DNA sample was added 1/10 volume of 3M sodiumacetate, pH 7.5 and 3 volumes of cold ethanol. The DNA was precipitatedfor 30 minutes at -70° C. or overnight at -20° C. and then pelleted bycentrifugation in the microfuge for 15 minutes at 4° C. The pellet waswashed once with 200 microliters of cold 80% ethanol and pelleted againfor 10 minutes at 4° C. After air drying or lyophilization, the pelletswere resuspended in the appropriate buffer or water.

RESTRICTION ENZYME DIGESTION. DNA was cut by restriction enzymes usingthe buffer recommended by the manufacturer (IBI, BRL, New EnglandBiolabs, etc). Whenever possibile, the concentration of DNA was keptbelow 1 microgram/50 microliters. Incubation was at 37° C. for 1-4hours.

AGAROSE GEL ELECTROPHORESIS OF DNA. To visualize the restriction patternof the DNA, 5 microliters of loading buffer (5X electrophoresis buffer,0.01% bromphenol blue dye, 50 mM EDTA, and 50% glycerol) were added. Thesample was loaded into a lane in a horizontal submarine electrophoresisunit containing a 0.6% to 3.0% agarose gel. The electrophoresis bufferwas 40 mM Tris, 10 mM EDTA, adjusted to pH 7.8 with acetic acid, andwith or without 0.5 micrograms/ml ethidium bromide. The gel was run at40-50 volts for 18 hours, removed and stained with 0.5 micrograms/mlethidium bromide for 30 minutes, and the DNA bands were visualized on along wavelength UV transilluminator.

LIGATION. DNA was joined together by the action of the enzyme T4 DNAligase (BRL). Ligation reactions contained various amounts of DNA (from0.2 to 20 μg), 20 mM Tris pH 7.5, 10 mM MgCl₂, 10 mM dithiothreitol(DTT), 200 μM ATP and 20 units T4 DNA ligase in 10-20 μl final reactionvolume. The ligation proceeded for 3-16 hours at 15° C.

SOUTHERN BLOTTING OF DNA. Southern blots utilized the Nonradioactive DNALabeling and Detection Kit of Boehringer Mannheim. The manufacturer'srecommended procedures were followed.

DNA TRANSFECTION FOR GENERATING RECOMBINANT VIRUS. The method is basedupon the calcium phosphate procedure of Graham and Van der eb (1) withthe following modifications. Virus and/or Plasmid DNA were diluted to298 μl in 0.01M Tris pH 7.5, 1 mM EDTA. Forty μl 2M CaCl₂ was addedfollowed by an equal volume of 2X HEPES buffered saline (10 gN-2-hydroxyethyl piperazine N'-2-ethanesulfonic acid (HEPES), 16 g NaCl,0.74 g KCI, 0.25 g Na₂ HPO₄.2H₂ O, 2 g dextrose per liter H₂ O andbuffered with NaOH to pH 7.4). The mixture was then incubated on ice for10 minutes, and then added dropwise to an 80% confluent monolayer ofVeto cells growing in a 60 mm petri dish under 5 ml of medium (DME plus2% fetal bovine serum). The cells were incubated four hours at 37° C. ina humidified incubator containing 5% CO₂. The cells were then washedwith three 5 ml aliquots of 1XPBS (1.15 g Na₂ HPO₄, 0.2 g KH₂ PO₄, 0.8 gNaCl, 0.2 g KCl per liter H₂ O), and fed with 5 ml of medium (DME plus2% fetal bovine serum). The cells were incubated at 37° C. as above for3-7 days until cytopathic effect from the virus was 50-100%. Virus washarvested as described above for the preparation of virus stocks. Thisstock is referred to as a transfection stock and was subsequentlyscreened for recombinant virus by the BLUOGAL SCREEN FOR RECOMBINANTPRV.

HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT PRV. Thismethod relies upon the homologous recombination between PRV DNA andplasmid homology vector DNA which occurs in Vero cells co-transfectedwith these elements. From 0.1.-1.0 μg of plasmid DNA containing foreignDNA flanked by appropriate PRV cloned sequences (the homology vector)were mixed with approximately 0.3 μg of intact PRV DNA. The DNA arediluted to 298 μl in 0.01M Tris pH 7.5, 1 mM EDTA and transfected intoVero cells according to the DNA TRANSFECTION FOR GENERATING RECOMBINANTVIRUS (see above).

DIRECT LIGATION PROCEDURE FOR GENERATING RECOMBINANT PRV. Rather thanusing homology vectors and relying upon homologous recombination togenerate recombinant virus, we have also developed the technique ofdirect ligation to engineer PRY. In this instance, a cloned foreign genedid not require flanking PRY DNA sequences but only required that ithave restriction sites available to cut out the foreign gene fragmentfrom the plasmid vector. A compatible restriction enzyme was used to cutPRV DNA. A requirement of the technique was that the restriction enzymeused to cut the PRV DNA must cut at a limited number of sites, at leastone of the sites being a nonessential site. We have used XbaI, whichcuts PRV DNA in two places. We have also used HindIII which cuts PRV DNAin four places. Restriction sites previously introduced into PRV byother methods may also be used. The PRV DNA was mixed with a 30-foldmolar excess of plasmid DNA (typically 5 μg of virus DNA to 10 μg ofplasmid DNA), and the mixture was cut with the appropriate restrictionenzyme. The DNA mixture was phenol extracted and ethanol precipitated toremove restriction enzymes, and ligated together according to theligation procedure detailed above. The ligated DNA mixture was thenresuspended in 298 μ1 0.01M Tris pH 7.5, 1 mM EDTA and transfected intoVero cells according to the DNA TRANSFECTION FOR GENERATING RECOMBINANTVIRUS (see above).

The direct ligation procedure may also be used to delete DNA from PRY.Non-essential DNA which is flanked by appropriate restriction enzymesites may be deleted by digesting the PRV with such enzymes andreligation. The frequency of engineered viruses generated by the directligation procedure is high enough that screening can be accomplished byrestriction enzyme analysis of randomly picked plaques from thetransfection stock.

BLUOGAL SCREEN FOR RECOMBINANT PRV. When the E. coli β-galactosidasemarker gene was incorporated into a recombinant virus the plaquescontaining recombinants were visualized by a simple assay. The chemicalBLUOGAL™ (halogenated indolyl-B-D-galactoside)(Bethesda Research Labs)was incorporated (200 μg/ml) into the agarose overlay during the plaqueassay, and plaques that expressed active β-galactosidase turned blue.The blue plaques were then picked onto fresh Vero cells and purified byfurther blue plaque isolations. In recombinant virus strategies in whichthe E. coli β-galactosidase marker gene is removed the assay involvesplaque purifying white plaques from a background of parental blueplaques. In both cases viruses were typically purified with three roundsof plaque purification.

CONSTRUCTION OF DELETION VIRUSES. The strategy used to constructdeletion viruses involved the use of both homologous recombination anddirect ligation techniques. Initially, a virus was constructed viahomologous recombination in which the gene to be deleted was replacedwith the E. coli β-galactosidase marker gene. A second virus was thenconstructed via direct ligation in which the marker gene was deleted.This strategy requires that the marker gene introduced into the firstvirus must be flanked by appropriate restriction enzyme sites asdescribed in the DIRECT LIGATION PROCEDURE FOR GENERATING RECOMBINANTPRV. The advantage of this strategy is that both viruses may be purifiedby the BLUOGAL SCREEN FOR RECOMBINANT PRV. The first virus is purifiedby picking blue plaques from a white background, the second virus ispurified by picking white plaques from a blue plaque background. Threedifferent homology vectors were constructed for the purpose of deletingthe gpX and gpI gene coding regions. A detailed description of thesehomology vectors follows.

HOMOLOGY VECTOR 263-58.18. The plasmid 263-58.18 was constructed for thepurpose of deleting the gpX gene coding region from the pseudorabiesvirus. It incorporates an E. coli β-galactosidase marker gene flanked byPRV DNA. Upstream of the marker gene is an approximately 1330 base pairfragment of PRV DNA which ends with sequences encoding the first sevenamino acids (4) of the gpX primary translation product. Downstream ofthe marker gene is an approximately 1800 base pair fragment of PRV DNAwhich begins with sequences encoding the last 19 amino acids (4) of thegpX primary translation product. When this plasmid is used according tothe HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT PRV itwill replace the DNA coding for amino acids 8-479 of the gpX primarytranslation product with DNA coding for the marker gene. Note that theβ-galactosidase (lacZ) marker gene will be under the control of theendogenous gpX promoter. A detailed description of the plasmid is givenin FIGS. 2A-2D. It was constructed from the indicated DNA sourcesutilizing standard recombinant DNA techniques (2). It may be constructedby joining restriction fragments from the following sources with thesynthetic DNA sequences indicated in FIGS. 2A-2D. The plasmid vector isderived from an approximately 2792 base pair SmaI to PvuII restrictionfragment of pSP65 (Promega). Fragment 1 is an approximately 922 basepair EcoRV to SalI restriction fragment of the PRY BamHI restrictionfragment #10 (15). Fragment 2 is an approximately 412 base pair SalI toBamHI restriction fragment of the PRV BamHI restriction fragment #10(15). Fragment 3 is an approximately 3347 base pair BamHI to Balirestriction fragment of plasmid pJF751 (11). Fragment 4 is anapproximately 1803 base pair NdeI to StuI restriction fragment from thePRV BamHI restriction fragment #7 (15). Note that the XbaI sites locatedat Junction B and Junction D are the only XbaI sites in this plasmid andpermit the marker gene to be cut out as an XbaI restriction fragment.

HOMOLOGY VECTOR 416-09.2H. The plasmid 416-09.2H was constructed for thepurpose of deleting the gI gene coding region from the pseudorabiesvirus. It incorporates an E. coli β-galactosidase marker gene flanked byPRV DNA. Upstream of the marker gene is an approximately 1884 base pairfragment of PRV DNA which ends with sequences located approximately 46base pairs upstream of the first amino acid (6) of the gI primarytranslation product. Downstream of the marker gene is an approximately1309 base pair fragment of PRV DNA which begins with sequences encodingthe last 105 amino acids (6) of the gI primary translation product. Whenthis plasmid is used according to the HOMOLOGOUS RECOMBINATION PROCEDUREFOR GENERATING RECOMBINANT PRV, it will replace the DNA coding for aminoacids 1-472 of the gI primary translation product with DNA coding forthe marker gene. Note that the β-galactosidase (lacZ) marker gene willbe under the control of the gpX promoter. A detailed description of theplasmid is given in FIGS. 3A-3D. It was constructed from the indicatedDNA sources utilizing standard recombinant DNA techniques (2). It may beconstructed by joining restriction fragments from the following sourceswith the synthethic DNA sequences indicated in FIGS. 3A-3D. The plasmidvector is derived from an approximately 3009 base pair SmaI to BamHIrestriction fragment of pSP19 (Promega). Fragment 1 is an approximately1884 base pair HincII to DraI restriction fragment of the PRV BamHIrestriction fragment #7 (15). Fragment 2 is an approximately 412 basepair SalI to BamHI restriction fragment of the PRV BamHI restrictionfragment #10 (15). Fragment 3 is an approximately 3347 base pair BamHIto Bali restriction fragment of plasmid pJF751 (11). Fragment 4 is anapproximately 1309 base pair SphI to BamHI restriction fragment from thePRV BamHI restriction fragment #7 (15). Note that the XbaI sites locatedat Junction B and Junction D are the only XbaI sites in this plasmid andpermit the marker gene to be cut out as an XbaI restriction fragment.

HOMOLOGY VECTOR 436-86.32K. The plasmid 436-86.32K was constructed forthe purpose of deleting the gpX gene coding region from the pseudorabiesvirus. It incorporates an HCMV immediate early promoted E. coliβ-galactosidase marker gene flanked by PRV DNA. Upstream of the markergene is an approximately 1330 base pair fragment of PRV DNA which endswith sequences encoding the first seven amino acids (4) of the gpXprimary translation product. Downstream of the marker gene is anapproximately 1800 base pair fragment of PRV DNA which begins withsequences encoding the last 19 amino acids (4) of the gpX primarytranslation product. When this plasmid is used according to theHOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT PRV, itwill replace the DNA coding for amino acids 8-479 of the gpX primarytranslation product with DNA coding for the marker gene. A detaileddescription of the plasmid is given in FIGS. 4A-4D. It was constructedfrom the indicated DNA sources utilizing standard recombinant DNAtechniques (2). It may be constructed by joining restriction fragmentsfrom the following sources with the synthetic DNA sequences indicated inFIG. 4. The plasmid vector is derived from an approximately 2792 basepair SmaI to PvuII restriction fragment of pSP65 (Promega). Fragment 1is an approximately 1336 base pair EcoRV to BamHI restriction fragmentof the PRV BamHI restriction fragment #10 (15). Fragment 2 is anapproximately 1191 base pair PstI to AvaII restriction fragment of theHCMV XbaI restriction fragment E (12). Fragment 3 is an approximately3002 base pair BamHI to PvuII restriction fragment of plasmid pJF751(11). Note that this fragment contains two internal PvuII sites,requiring a partial PvuII digest. Alternatively, the fragment may beobtained as two fragments without the requirement of a partial digest.These two fragments are an approximately 2950 base pair BamHI to NdeIfragment and an approximately 55 base pair NdeI to PvuII fragment. Thesetwo fragments joined at the unique NdeI site may substitute for fragment3. Fragment 4 is an approximately 1803 base pair NdeI to StuIrestriction fragment from the PRY BamHI restriction fragment #7 (15).Note that the XbaI sites located at Junction B and Junction D are theonly XbaI sites in this plasmid and permit the marker gene to be cut outas an XbaI restriction fragment.

VACCINATION STUDIES IN SWINE. Three-day-old pigs born to sows from swineherds free of pseudorabies were used to test the efficacy of the live,attenuated virus. The piglets were inoculated intramuscularly with 1 mlof virus fluid containing about 10³ to 10⁵ plaque forming units (PFU).

PRV-155 virus was also tested as an inactivated vaccine. The virus wasinactivated by exposure to binary ethyleneimine for at least 40 hours at37° C. Inactivated virus fluids were blended with an oil-based adjuvantand inoculated into four-week-old pigs.

Animals vaccinated with either live S-PRV-155 or with inactivatedS-PRV-155 were observed each day after vaccination for adverse reactions(clinical signs of PRV disease). Samples of nasal secretions wereobtained from pigs vaccinated with live S-PRV-155 and cultured todetermine if the vaccine virus was capable of shedding and spreading toother animals. Immunity was determined by measuring PRV serum antibodylevels and by challenging the vaccinated pigs with virulent virus at 3-4weeks post-vaccination. In the latter case, the vaccinated animals and agroup of non-vaccinated animals were inoculated with a virulent,pneumotropic strain of PRV (VDL4892), using an amount of challenge virusthat caused PRY disease in at least 80% of the unvaccinated group ofpigs. The challenged animals were observed daily for signs of diseaseand for nasal virus shedding. Serum samples were obtained at the time ofchallenge and at weekly intervals for 2-3 weeks post-vaccination. Theserum was assayed for serum neutralizing (SN) antibody and for antibodyto gpX (HERDCHEK® (in vitro diagnostic test for detection of PRV gpX andgI antibodies, IDEXX corp.) and gpI with (HERDCHEK® (in vitro diagnostictest for detection of PRV gpX and gI antibodies, IDEXX Corp.) andCLINEASE® (in vitro diagnostic test for detection of PRV gI antibodies),SmithKline Beecham) according to manufacturer's recommendations.

EXAMPLES Example 1

S-PRY-150

S-PRV-150 is a pseudorabies virus that has a deletion in the TK gene inthe long unique region, a deletion in the repeat region, a 1460 basepair deletion in the gI coding region, and a 1825 base pair deletion inthe gpX coding region. The gene for E. coli β-galactosidase (lacZ gene)was inserted in the place of the gPI gene and is under the control ofthe HCMV immediate early promoter.

S-PRV-150 was derived from S-PRV-002 (U.S. Pat. No. 4,877,737, issuedOct. 31, 1989) through the construction of three intermediate viruses.The first intermediate virus was S-PRV-070. In this virus the XbaI siteslocated in the repeat regions (see FIG. 1) were converted to EcoRI sitesto allow for the use of XbaI restriction sites at other locations insubsequent steps. This was accomplished by inserting the syntheticoligonucleotide CTAGGAATTCC into the XbaI sites of S-PRV-002 utilizingthe DIRECT LIGATION PROCEDURE FOR GENERATING RECOMBINANT PRV. In thesecond intermediate virus S-PRV-089, the gpX deletion was introducedalong with the β-galactosidase marker gene. This was accomplishedutilizing the homology vector 263-58.18 (see Materials and Methods) andvirus S-PRV-070 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATINGRECOMBINANT PRY. In the third intermediate virus (S-PRV-112) theβ-galactosidase marker gene (lacZ) was removed by digestion with XbaI asdescribed in the DIRECT LIGATION PROCEDURE FOR GENERATING RECOMBINANTPRV. Finally, S-PRV-150 was generated utilizing the homology vector416-09.2H (see Materials and Methods) and virus S-PRV- 153 in theHOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT PRV. Thestructure of S-PRV-150 was confirmed by restriction enzyme analysis withBamHI and XbaI.

Example 2

S-PRV-151

S-PRV-151 is a pseudorabies virus that has a deletion in the TK gene inthe long unique region, a deletion in the repeat region, a 1460 basepair deletion in the gI coding region, and a 1825 base pair deletion inthe gpX coding region.

S-PRV-151 resulted from the removal of the marker gene from S-PRV-150(see above). This was accomplished by digestion of S-PRY-150 with XbaIas described in the DIRECT LIGATION PROCEDURE FOR GENERATING RECOMBINANTPRV. The structure of S-PRV-151 was confirmed by restriction enzymeanalysis with BamHI and XbaI.

The following experiment was conducted to determine if S-PRY-151 may beused as a vaccine to protect swine against pseudorabies disease. In thisstudy, three-day-old piglets were vaccinated intramuscularly withS-PRV-151 as follows: 4 were inoculated with 10⁶ PFU and 4 wereinoculated with 10⁶ PFU. A control group of 5 was also included in thestudy. The animals were observed, then challenged as described inVACCINATION STUDIES WITH SWINE (see Table I below).

                                      TABLE I                                     __________________________________________________________________________    VACCINATION OF 3-DAY-OLD PIGLETS WITH S-PRV-151                               AND CHALLENGE WITH VIRULENT PRV                                                          SN Antibody.sup.a                                                  Virus  No. of                                                                            Day 21 Post                                                                          Day 14 Post                                                                          Post Challenge Observations                          Conc.  pigs                                                                              Vaccination                                                                          Challenge                                                                            Virus Shedding.sup.b                                                                   Clinical Signs.sup.c                                                                  Death                               __________________________________________________________________________    10.sup.4 PFU per                                                                     4   ≦2                                                                            46     41%      100%    20%                                 Dose                                                                          10.sup.6 PFU per                                                                     4   ≦2                                                                            96     33%      100%    0                                   Dose                                                                          None   6   <2     10     55%      100%    20%                                 __________________________________________________________________________     .sup.a Geometric mean titer (reciprocal of dilution)                          .sup.b Percent of nasal secretion samples positive for virus                  .sup.c Percent of pigs with CNS and/or respiratory manifestation         

In this experiment, all of the vaccinated animals remained healthyfollowing vaccination, and did not shed vaccine virus in tonsillarsecretions; however, neutralizing antibodies were not detected. Afterchallenge with virulent virus, all vaccinates from both groups exhibitedclinical signs of PRY disease. Although S-PRV-151 was shown to be safein three-day-old piglets, this virus failed to demonstrate anyprotection in these animals.

Example 3

S-PRV-154

S-PRV-154 is a pseudorabies virus that has a deletion in the TK gene inthe long unique region, a deletion in the repeat region, a 1460 basepair deletion in the gI coding region, and a 1414 base pair deletion inthe gpX coding region. The gene for E. coli β-galactosidase (lacZ gene)was inserted in the place of the gpX gene and is under the control ofthe HCMV immediate early promoter.

S-PRV-154 was derived from S-PRV-002 (U.S. Pat. No. 4,877,737, issuedOct. 31, 1989) through the construction of three intermediate viruses.The first intermediate virus was S-PRV-070. In this virus, the XbaIsites located in the repeat regions (see FIG. 1) were converted to EcoRIsites to allow for the use of XbaI restriction sites at other locationsin subsequent steps. This was accomplished by inserting the syntheticoligonucleotide CTAGGAATTCC into the XbaI sites of S-PRV-002 utilizingthe DIRECT LIGATION PROCEDURE FOR GENERATING RECOMBINANT PRV. In thesecond intermediate virus S-PRV-146, the gI deletion was introducedalong with the β-galactosidase marker gene. This was accomplishedutilizing the homology vector 416-09.2H (see Materials and Methods) andvirus S-PRV-070 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATINGRECOMBINANT PRV. In the third intermediate virus (S-PRV-153) theβ-galactosidase marker gene was removed by digestion with XbaI asdescribed in the DIRECT LIGATION PROCEDURE FOR GENERATING RECOMBINANTPRV. Finally, S-PRV-154 was generated utilizing the homology vector 43686.32K (see Materials and Methods) and virus S-PRV-153 in the HOMOLOGOUSRECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT PRV. The structure ofS-PRV-154 was confirmed by restriction enzyme analysis with BamHI andXbaI.

Example 4

S-PRV-155

S-PRV-155 is a pseudorabies virus that has a deletion in the TK gene inthe long unique region, a deletion in the repeat region, a 1460 basepair deletion in the gI coding region, and a 1414 base pair deletion inthe gpX coding region. The S-PRV-155 pseudorabies virus has beendeposited pursuant to the Budapest Treaty on the International Depositof Microorganisms for the Purposes of Patent Procedure with the PatentCulture Depository of the American Type Culture Collection, 12301Parklawn Drive, Rockville, Md. 20852 U.S.A. under ATCC Accession No. VR2311.

S-PRY-155 resulted from the removal of the β-galactosidase marker genefrom S-PRY-154 (see above). This was accomplished by digestion ofS-PRV-154 with XbaI as described in the DIRECT LIGATION PROCEDURE FORGENERATING RECOMBINANT PRV. The structure of S-PRV-155 was confirmed byrestriction enzyme analysis with BamHI and XbaI. The gpX and gIdeletions were confirmed by Southern blot analysis with probes for thecoding regions of each gene.

The following experiments indicate that S-PRV-155 is useful as a vaccineto protect swine against pseudorabies disease and that it produces animmune response which can be distinguished from wild-type infection.

In this study, three-day-old piglets were vaccinated intramuscularlywith S-PRV-155 as follows: 6 were inoculated with 10³ PFU, 8 wereinoculated with 10⁵ PFU, and 20 were inoculated with 10³.5 PFU of virus.Two control groups of 5 each were also included in the study. Theanimals were observed, then challenged as described in VACCINATIONSTUDIES WITH SWINE (see Table II below).

                                      TABLE II                                    __________________________________________________________________________    VACCINATION OF 3-DAY-OLD PIGLETS WITH S-PRV-155                               AND CHALLENGE WITH VIRULENT PRV                                                                                              Post-Challenge                                                                Observations                             SN Antibody.sup.a                                                                           Post-Vaccination                                                                         Post-Challenge                                                                            Virus                          Virus No. of                                                                            Post          Diagnostic Antibody.sup.b                                                                Diagnostic Antibody                                                                       Shed-                                                                              Clinical                  Conc. Pigs                                                                              Vaccination                                                                         Post Challenge                                                                        gpX.sup.c                                                                         gpl.sup.c                                                                        gpl.sup.d                                                                         gpX.sup.c                                                                         gpl.sup.c                                                                         gpl.sup.d                                                                         ding.sup.g                                                                         Signs                                                                              Death                __________________________________________________________________________    10.sup.3 pfu per                                                                    6   7     50      0   0  0   67%.sup.c                                                                         NT   16%                                                                              21%  0    0                    Dose                                                                          10.sup.5 pfu per                                                                    8   5     60      0   0  0    62%                                                                              NT   37%                                                                              12%  0    0                    Dose                                                                          None  5   <2    25      0   0  0   100%                                                                              NT  100%                                                                              50%  100% 0                    10.sup.3.5 pfu                                                                      20  4     316     0   0  0   90%.sup.f                                                                         100%                                                                              100%                                                                              34%  0    0                    per Dose                                                                      None  5   <2    24      0   0  0   100%                                                                              100%                                                                              100%                                                                              73%  100%  80%                 __________________________________________________________________________     .sup.a Geometric mean titer (reciprocal of dilution)                          .sup.b Day of challenge?                                                      .sup.c HerdChek test; percent positive                                        .sup.d ClinEase test; percent positive                                        .sup.e Day 14 postchallenge                                                   .sup.f Day 21 postchallenge                                                   .sup.g Percent of nasal secretion samples positive for virus             

In this experiment, all of the vaccinated animals remained healthyfollowing vaccination, developed serum neutralizing antibody to PRV anddid not shed vaccine virus in tonsillar secretions. After challenge withvirulent virus, vaccinates of all three groups remained free of PRVdisease, whereas all animals in the control groups developed clinicalsigns of PRV disease, including 80% death in one group.

The serum samples collected from the vaccinated and challenged animalswere assayed by the gX HerdChek test rand by the gI HERDCHEK® (in vitrodiagnostic test for detection of PRV gpX and gI antibodies) andCLINEASE® (in vitro diagnostic test for detection of PRV gI antibodies)tests. As expected the swine vaccinated with S-PRV-155 remainsero-negative gpX and gI up to the day of challenge. The vaccinatedanimals were protected by the vaccination from pseudorabies disease whenchallenged with the wild-type virus. However, vaccinated animals wereasymptomatically super-infected by the challenge strain and would,therefore, be expected to produce antibodies to gpX and gI uponchallenge.

As shown in Table II, serum from animals vaccinated with S-PRV-155remained negative for gpX and gI until after challenge with wild-typevirus. By 14 days post-challenge, vaccinates had began seroconverting togpX and gI. These results indicate that S-PRV-155 is an effectivevaccine strain which permits vaccinates to be distinguished from animalsinfected with wild-type virus by a simple serum diagnostic assay.

Example 5

The following experiment indicates that inactivated S-PRV-155 may beused as a vaccine to protect swine against pseudorabies disease and thatit produces an immune response which can be distinguished from wild-typeinfection. Five young, seronegative pigs were vaccinated once with aninactivated S-PRV-155 vaccine and challenged as described in VACCINATIONSTUDIES IN SWINE. The inactivated S-PRV-155 vaccine had a virusconcentration of 10⁷.33 PFU/dose (see Table III below).

                                      TABLE III                                   __________________________________________________________________________    VACCINATION OF YOUNG PIGS WITH INACTIVATED                                    S-PRV-155 AND CHALLENGE WITH VIRULENT PRV                                                                                 POST-CHALLENGE                                 SN ANTIBODY.sup.a                                                                           DIAGNOSTIC ANTIBODY.sup.b                                                                      OBSERVATIONS                               NO. POST-         POST-    POST-   VIRUS                                                                              CLIN-                                 OF  VACCI-                                                                              POST-   VACCINATION                                                                            CHALLENGE                                                                             SHED-                                                                              ICAL                         GROUP    PIGS                                                                              NATION                                                                              CHALLENGE                                                                             gX  g1   gX  g1  DING.sup.c                                                                         SIGNS.sup.d                                                                           DEATH.sup.d          __________________________________________________________________________    VACCINATES                                                                             5   19    170     0   0     80 100 15    20     0                    CONTROLS 5   <2     32     0   0    100 100 75   100     0                    __________________________________________________________________________     .sup.a Geometric mean titer expressed as reciprocal of dilution               .sup.b Percent seroconverting                                                 .sup.c Percent of nasal secretion samples positive for virus                  .sup.d Percent of animals                                                

Vaccinates developed serum neutralizing antibody to PRV followingvaccination, but not to gpX or gI. After challenge with virulent virus,one vaccinate showed incoordination for one day only. All 5 unvaccinatedcontrols developed severe central nervous system manifestations orpseudorabies disease. At least 21 days after challenge, the vaccinateshad seroconverted to one or both diagnostic antigens. These resultsindicate that S-PRV-155, in an inactivated vaccine, protects swineagainst pseudorabies disease and produces an immune response that can bedistinguished from that of infected animals.

Example 6

S-PRV-158

S-PRV-158 is a pseudorabies virus that has a 1460 base pair deletion inthe gI coding region, and a 1414 base pair deletion in the gpX codingregion. The gene for E. coli β-galactosidase (lacZ gene) was inserted inthe place of the gpI gene and is under the control of the HCMV immediateearly promoter.

S-PRV-158 was derived from S-PRV-000 (PRV strain ISU S62/26) through theconstruction of two intermediate viruses. The first intermediate viruswas S-PRV-156. In the first intermediate virus S-PRV-156, the gpXdeletion was introduced along with the β-galactosidase (lacZ) markergene. This was accomplished utilizing the homology vector 436-86.32K(see Materials and Methods) and virus S-PRV-000 in the HOMOLOGOUSRECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT PRV. In the secondintermediate virus (S-PRY-157) the β-galactosidase marker gene (lacZ)was removed by digestion with XbaI as described in the DIRECT LIGATIONPROCEDURE FOR GENERATING RECOMBINANT PRV. Finally, S-PRV-158 wasgenerated utilizing the homology vector 416-09.2H (see Materials andMethods) and virus S-PRV-157 in the HOMOLOGOUS RECOMBINATION PROCEDUREFOR GENERATING RECOMBINANT PRY. The structure of S-PRV-158 was confirmedby restriction enzyme analysis with BamHI and XbaI.

Example 7

S-PRY-159

S-PRV-159 is a pseudorabies virus that has a 1460 base pair deletion inthe gI coding region and a 1414 base pair deletion in the gpX codingregion.

S-PRV-159 resulted from the removal of the marker gene from S-PRV-158(see above). This was accomplished by digestion of S-PRV-158 with XbaIas described in the DIRECT LIGATION PROCEDURE FOR GENERATING RECOMBINANTPRV. The structure of S-PRV-159 was confirmed by restriction enzymeanalysis with BamHI and XbaI.

Summary of Examples

The present invention involves the use of genetically engineeredherpesviruses to protect animals against disease. These virusesincorporate the deletion of two glycoproteins, gpX and gI. Table IVsummarizes characteristics of the six deletion viruses described here.Virus titers were determined according to the TITRATION OF PRV. Thesafety and efficacy data were generated according to the VACCINATIONSTUDIES IN SWINE.

                  TABLE IV                                                        ______________________________________                                        CHARACTERISTICS OF PRV gpX gI DELETION VIRUSES                                                     Post Challenge                                                                Observations                                                     Titer     Post Vaccination                                                                           Clinical                                       Virus # pfu/ml    Clinical Signs                                                                             Signs   Death                                  ______________________________________                                        S-PRV-150                                                                             2.30 × 10.sup.6                                                                   NT           NT      NT                                     S-PRV-151                                                                             3.80 × 10.sup.5                                                                   NONE         100%    20%                                    S-PRV-154                                                                             1.53 × 10.sup.8                                                                   NT           NT      NT                                     S-PRV-155                                                                             1.48 × 10.sup.8                                                                   NONE          0%      0%                                    S-PRV-158                                                                             1.47 × 10.sup.8                                                                   NT           NT      NT                                     S-PRV-159                                                                             5.61 × 10.sup.8                                                                   NT           NT      NT                                     ______________________________________                                    

All six viruses incorporate deletions of gpX and gI. The region of thegpX deletion in S-PRV-150 and S-PRV-151 is larger than the gpX deletionin the other viruses. Viruses S-PRV-150, S-PRV-151, S-PRV-154, andS-PRV-155 also have deletions in the TK and the repeat regions. TheE-coli βB-galactosidase (lacZ) marker gene is present in virusesS-PRV-150, S-PRV-154, and S-PRV-158.

From this analysis it is clear that the precise size and combination ofdeletions is important to the efficacy of a differential vaccineincorporating both gpX and gI markers. S-PRV-150 and S-PRV-151 exhibitsignificantly reduced virus titers. The titer to which virus may begrown in cell culture is critical to the manufacture of vaccine. The lowtiters of S-PRV-150 and S-PRV-151 would make their manufacture asvaccines cost prohibitive. S-PRV-155 and S-PRV-151 also differdramatically in their ability to protect swine from PRV disease.S-PRV-151 provides an unacceptable level of protection, whereasS-PRV-155 provides complete protection. The combination of deletionspresent in S-PRV-155 is responsible for making this virus strain asuperior vaccine product. This product provides safety in three-day-oldpiglets, protection in all age pigs, and serologic differentiation usingeither one or two diagnostic markers. Based on the current state of theart, the superior utility of this combination of deletions wasunexpected. These results are novel, unpredictable, and useful in theselection of a superior pseudorabies vaccine product.

References

1. F. L. Graham and A. Van der Eb., Virology 52, 556-567, 1973.

2. T. Maniatis, et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Press, 1982.

3. M. van Zijl, et al., J. of Virology 71, 1747-1755, 1990.

4. T. J. Rea, et al., J. of Virology 54, 21-29, 1985.

5. E. A. Petrovskis, et al., J. of Virology 59, 216-223, 1986.

6. E. A. Petrovskis, et al., J. of Virology 60, 185-193, 1986.

7. E. A. Petrovskis, et al., J. of Virology 60, 1166-1169, 1986.

8. T. Ben-Porat and A. S. Kaplan, Virology 41, 265-273, 1970.

9. T. Ben-Porat, et al., J. of Virology 49, 970-979, 1984.

10. T. Ben-Porat, et al., Virology 154, 325-334, 1986.

11. F. A. Ferrari, et al., J. of Bacteriology 161, 556-562, 1985.

12. D. R. Thomsen, et al., Gene 16, 207-216, 1981.

13. R. W. Price and A. Kahn, Infection and Immunity 34, 571-580, 1981.

14. P. B. Tenser, et al., J. of General Virology 64, 1369-1373, 1983.

15. B. Lomniczi, et al., J. of Virology 49, 970-979, 1984.

16. B. Roizman, et al., Cold Spring Harbor Conference on New Approachesto Viral Vaccines, September, 1983.

17. R. L. Thompson, et al., Virology 131, 180-192, 1983.

18. K. Fukuchi, et al., Proc. Natl. Acad. Sci. U.S.A. 82, 751-754, 1985.

9. J. M. Koomey, et al., J. of Virology 50, 662-665, 1984.

20. M. W. Ligas and D. C. Johnson, J. of Virology 62, 1486-1494, 1988.

21. F. Zuckerman, et al., Vaccination and Control of Adjeszky's Disease,pp. 107-117, Ed. J. van Oirschot. Kluwer, London, 1989.

22. T. C. Mettenleiter, et al., Virology 158, 141-146, 1987.

23. A. K. Robbins, et al., J. of Virology 58, 339-347, 1986.

24. A. K. Robbins, et al., J. of Virology 61, 2691-2701, 1987.

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    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 32                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 57 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: N                                                         (iv) ANTI-SENSE: N                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       TATAGAATACACGGAATTCGAGCTCGCCCATCGCGGCACGCCGGCCGTCCCGGCGCT57                   (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 57 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: DNA (genomic)                                              (iii) HYPOTHETICAL: N                                                        (iv) ANTI-SENSE: N                                                            (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..57                                                           (D) OTHER INFORMATION:                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       GCGCGCGACGCGTACGACACCAAGGTCGACTCTAGAGTCGACGTCTGG48                            AlaArgAspAlaTyrAspTh rLysValAspSerArgValAspValTrp                             151015                                                                        GGCGCGGGG57                                                                   GlyAlaGly                                                                     (2) INFORMATION FOR SEQ ID NO:3:                                               (i) SEQUENCE CHARACTERISTICS:                                                (A) LENGTH: 19 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       AlaArgAspAlaTyrAspThrLysValAspSerArgValAspValTrp                              1510 15                                                                       GlyAlaGly                                                                     (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 57 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: N                                                         (iv) ANTI-SENSE: N                                                            (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                              (B) LOCATION: 13..57                                                         (D) OTHER INFORMATION:                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       CACAGCTCAACAATGAAGTGGGCAACGTGGATCGATCCCGTCGTTTTA48                            MetLysTrpAlaThrTrpIleAspProValValLeu                                           1510                                                                         CAACGTCGT57                                                                   GlnArgArg                                                                     15                                                                            (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       MetLysTrpAlaThrTrpIleAspProValValLeuGlnArgArg                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 107 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: N                                                         (iv) ANTI-SENSE: N                                                            (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..9                                                            (D) OTHER INFORMATION:                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                              (B) LOCATION: 48..104                                                        (D) OTHER INFORMATION:                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       AAACTCGGGCAGCGTTGGGTCCTGGGACTCTAGAGGATCGATCCCCTATGGCG53                       LysLeuGlyMetAla                                                               1 1                                                                           ATCATCAGGGCCCGGGCCCGGAACGACGGCTACCGCCACGTGGCCTCC101                           IleIleArgAlaArgAlaArgAsnAspGlyTyrArgHisValAlaSer                              5 1015                                                                        GCCTGA107                                                                     Ala                                                                           (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 3 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                      LysLeuGly                                                                     (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       MetAlaIleIleArgAlaArgAlaArgAsnAspGlyTyrArgH isVal                             151015                                                                        AlaSerAla                                                                     (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 107 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: DNA (genomic)                                             (i ii) HYPOTHETICAL: N                                                        (iv) ANTI-SENSE: N                                                            (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..9                                                            (D) OTHER INFORMATION:                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 48..104                                                         (D) OTHER INFORMATION:                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       AAACTCGGGCAGCGTTGGGTCCTGGGACTCTA GAGGATCGATCCCCTATGGCG53                      LysLeuGlyMetAla                                                               11                                                                            ATCATCAGGGCCCGGGCCCGGAACGACGGCTAC CGCCACGTGGCCTCC101                          IleIleArgAlaArgAlaArgAsnAspGlyTyrArgHisValAlaSer                              51015                                                                         GCCTGA 107                                                                    Ala                                                                           (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 3 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      LysLeuGly                                                                     1                                                                             (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 amino acids                                                     (B) TYPE: amino acid                                                         (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      MetAlaIleIleArgAlaArgAlaArgAsnAspGlyTyrArgHisVal                              151015                                                                        AlaSerAla                                                                      (2) INFORMATION FOR SEQ ID NO:12:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 57 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: N                                                         (iv) ANTI-SENSE: N                                                            (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..33                                                           (D) OTHER INFORMATION:                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                     GAGTACGACCTCTGCCCCCGCGTGCACCACGAGGCTGCATTAATGAATCGGCC53                       GluTyrAspLeuCysProArgValHisHisGlu                                             1510                                                                          AACG 57                                                                       (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      GluTyrAspLeuCysProArgValHisHisGlu                                              1510                                                                         (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 57 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: N                                                         (iv) ANTI-SENSE: N                                                            (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                            (B) LOCATION: 31..57                                                          (D) OTHER INFORMATION:                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      ACCATGATTACGAATTCGAGCTCGGTACCCGACGGCGTGAACATCCTCACCGAC54                      AspGlyValAsnIleLeuThrAsp                                                       15                                                                           GAC57                                                                         Asp                                                                           (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          ( D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      AspGlyValAsnIleLeuThrAspAsp                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 57 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: N                                                         (iv) ANTI-SENSE: N                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      TTATCTCCGTCCGCGCCGTTTTGGGGATCCTCTAGAGTCGACGTCTGGGGCGCGGGG57                   (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 57 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: circular                                                       (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: N                                                         (iv) ANTI-SENSE: N                                                            (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 13..57                                                          (D) OTHER INFORMATION:                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      CACAGCTCAACAATGAAGTGGGCAACGTGGATCGATCCCGTCGTTTTA 48                           MetLysTrpAlaThrTrpIleAspProValValLeu                                          1510                                                                          CAACGTCGT57                                                                   GlnA rgArg                                                                    15                                                                            (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      MetLysTrpAlaThrTrpIleAspProValValLeuGlnArgArg                                 1 51015                                                                       (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 108 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: N                                                         (iv) ANTI-SENSE: N                                                            (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..21                                                           (D) OTHER INFORMATION:                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 61..108                                                         (D) OTHER INFORMATION:                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      CTGCTTTCTGACAAACTCGGGCAGCGTTGGGTCCTGGGACTCTAGAGTCGA5 1                        LeuLeuSerAspLysLeuGly                                                         15                                                                            CCTGCAGGCATGCTCTCGCCGGTGTACACCAGCCTGCCCACGCACGAG99                            MetLeuSerProValTyrThrSerLeuProThrHisGlu                                       1 510                                                                         GACTACTAC108                                                                  AspTyrTyr                                                                     15                                                                            (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                         (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      LeuLeuSerAspLysLeuGly                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                       MetLeuSerProValTyrThrSerLeuProThrHisGluAspTyrTyr                             151015                                                                        (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 57 base pairs                                                     (B) TYPE: nucleic acid                                                        ( C) STRANDEDNESS: double                                                     (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: N                                                         (iv) ANTI-SENSE: N                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      ACACGGAGTGGTCCTCCGTCATCAACGGGATCCCCGGGCGAGCTCGAATTCCGTGTA57                   (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 58 base pairs                                                      (B) TYPE: nucleic acid                                                       (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: N                                                         (iv) ANTI-SENSE: N                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      TATAGAATACACGGAATTCGAGCTCGCCCATCGCGGCACGCCGGCCGTCCCGGCGCTC58                  (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 58 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: N                                                         (iv) ANTI-SENSE: N                                                            (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 7..27                                                           (D) OTHER INFORMATION:                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      TCAACAATG AAGTGGGCAACGTGGATCGGGGATCCTCTAGAGTCGAC47                            MetLysTrpAlaThrTrpIle                                                         15                                                                            CTGCAGTGAAT58                                                                 (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      MetLysTrpAlaThrTrpIle                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 58 base pairs                                                      (B) TYPE: nucleic acid                                                       (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: N                                                         (iv) ANTI-SENSE: N                                                            (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 40..58                                                          (D) OTHER INFORMATION:                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      ATCCACGCTGTTTTGACCTCCATAGAA GACACCGGGACCATGGATCCCGTCGTT54                     MetAspProValVal                                                               15                                                                            TTAC 58                                                                       Leu                                                                           (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      MetAspProValValLeu                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:28:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 216 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: N                                                         (iv) ANTI-SENSE: N                                                            (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..84                                                           (D) OTHER INFORMATION:                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 134..190                                                        (D) OTHER INFORMATION:                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                      TGGAGCCCGTCAGTATCGGCGGAATTACAGCTGAGCGCCGGTCGCTAC48                            TrpSerProSerValSerAlaGluLeuGlnLeuSerA laGlyArgTyr                             151015                                                                        CATTACCAGTTGGTCTGGTGTCAAAAAGATCTAGAATAAGCTAGAG94                              HisTyrGlnLeuValTrpCysGlnLysAspLeuGlu                                           2025                                                                         GATCGATCCCCTATGGCGATCATCAGGGCCCGATCCCCTATGGCGATCATCAGG148                     MetAlaIleIleArg                                                                15                                                                           GCCCGGGCCCGGAACGACGGCTACCGCCACGTGGCCTCCGCC190                                 AlaArgAlaArgAsnAspGlyTyrArgHisValAlaSerAla                                    1015                                                                          TGACCCGGCCCCGCCCGACTCCCCCG216                                                 (2) INFORMATION FOR SEQ ID NO:29:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                      TrpSerProSerV alSerAlaGluLeuGlnLeuSerAlaGlyArgTyr                             151015                                                                        HisTyrGlnLeuValTrpCysGlnLysAspLeuGlu                                          2025                                                                          (2 ) INFORMATION FOR SEQ ID NO:30:                                            (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                      MetAlaIleIleArgAlaArgAlaArgAsnAspGlyTyrArgHisVal                              15 1015                                                                       AlaSerAla                                                                     (2) INFORMATION FOR SEQ ID NO:31:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 58 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: circular                                                        (ii) MOLECULE TYPE: DNA (genomic)                                             (iii) HYPOTHETICAL: N                                                         (iv) ANTI-SENSE: N                                                            (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                            (B) LOCATION: 1..33                                                           (D) OTHER INFORMATION:                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                      GAGTACGACCTCTGCCCCCGCGTGCACCACGAGGCTGCATTAATGAATCGGCC53                       GluTyrAspLeuCysProArgValHisHisGlu                                             15 10                                                                         AACGC58                                                                       (2) INFORMATION FOR SEQ ID NO:32:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                      GluTyrAspLeuCysProArgValHisHisGlu                                             1510                                                                      

What we claim is:
 1. An attenuated, genetically-engineered pseudorabiesvirus designated S-PRV-155 (ATCC Accession No. VR 2311).
 2. A method ofdistinguishing an animal vaccinated with a vaccine, said vaccinecomprising an attenuated, genetically-engineered pseudorabies virusdesignated S-PRV-155 (ATCC Accession No. VR 2311), from an animalinfected with a naturally-occurring, wild-type pseudorabies virus whichcomprises detecting the presence or absence of pseudorabies virusglycoprotein X in a body fluid of the animal.
 3. The method of claim 2,wherein the presence or absence of the glycoprotein X in the body fluidis determined by detecting in the body fluid antibodies specific for theglycoprotein X.
 4. The method of claim 2, wherein the method furthercomprises detecting the presence or absence of pseudorabies glycoproteinI in the body fluid of the animal.
 5. The method of claim 2, wherein theanimal is a swine.
 6. A method of distinguishing an animal vaccinatedwith a vaccine, said vaccine comprising an attenuated,genetically-engineered pseudorabies virus designated S-PRV-155 (ATCCAccession No. VR 2311), from an animal infected with anaturally-occurring, wild-type pseudorabies virus which comprisesdetecting the presence or absence of the glycoprotein I in a body fluidof the animal.
 7. The method of claim 6, wherein the presence or absenceof the glycoprotein I in the body fluid is determined by detecting inthe body fluid antibodies specific for the glycoprotein I.
 8. The methodof claim 6, wherein the animal is a swine.